Project Summary The abuse of opioids, which include heroin and synthetic opioids (e.g., fentanyl), is a growing problem in the United States that is partly responsible for the recently declared National Emergency for Opioid crisis. Between 2006 and 2015, the number of heroin-related deaths increased over 600% where heroin laced synthetic opioids (e.g., the ?killer opioids?) is responsible most of the deaths from abuse of opioids. Although FDA-approved medications to treat opioid addiction are available, the utilization rate for these medications is limited from side effects, tight prescription guidelines, and restricted availability. The ongoing rise in opioid abuse creates a dire need for new therapy that offers long-lasting, safe, and cost- effective interventions for overdoses and relapses but avoid side effects associated with current addiction medications. Immunopharmacotherapy using drug-specific antibodies (i.e., immunoantagonists) to block opioid drug activity and prevent the target opioids entering the brain are promising treatment approach that have less side-effect than the traditional drug-based opioid replacement treatment. In this SBIR project, we propose to develop a new class of immunotherapeutics-the nanobodies (e.g. the single domain antibodies, sdAbs) that have high specificities and affinities to heroin, fentanyl for the treatment of ?killer opioids? overdose and abuse. We expect the opioid-specific nanobodies will be better immunotherapeutics than traditional immunoglobulin G-based drug antibodies because nanobodies are extremely robust, highly resistant to denaturation, have superior tissue penetration, and have ability to cross blood brain barrier. Our goal is to achieve long-lasting and high titer antibodies to opioid drugs of abuse. The phase I project will focus on in vitro methodologies for the generation of specific nanobodies with high affinities to ?killer opioids? from phage-display nanobody library. In the Phase I project, we will make derivatives of opioid drugs such that they can be chemically conjugated at high density on magnetic beads, and isolate high affinity nanobodies specific to heroin and fentanyl. Furthermore, we will engineer and express individual single domain antibody to produce bivalent (multivalent) nanobody or multi- nanobody-conjugated nanoparticles as new therapeutic modalities targeting two or more opioids simultaneously. These nanobodies will improve upon current drug-specific antibody paradigms by increasing multivalency and allowing pharmacokinetic customization, while avoiding interactions with endogenous antibody receptor pathways. Successful completion of Phase I project will generate novel opioid-specific nanobodies and provide a framework for the development of immunotherapeutics against other drugs of abuse. In future Phase II, we will perform the pre-clinical in vitro and in vivo rodent pharmacokinetic study of individual nanobodies and multivalent nanobody for therapeutic neutralizations of the killer opioids.